Journal
ANALYTICAL CHEMISTRY
Volume 84, Issue 18, Pages 7633-7637Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac3017746
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Funding
- National Basic Research Program of China [2012CB910601]
- National Nature Science Foundation [21027002, 21175131]
- Creative Research Group Project by NSFC [21021004]
- Innovation Method Research of MOST [2010IM030500]
- Knowledge Innovation Project of Chinese Academy of Sciences
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Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, SO mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, alpha-2-macroglobulin, alpha-1-antitrypsin, apolipoprotein B-100, Ig gamma-2 chain C region, haptoglobin, hemopodn, alpha-1-acid glycoprotein 1, and alpha-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.
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